Sample information curated by ChIP-Atlas

Antigen

Antigen Class
TFs and others
Antigen
KDM1A

Cell type

Cell type Class
Kidney
Cell type
293
Primary Tissue
Kidney
Tissue Diagnosis
Normal

Attributes by original data submitter

Sample

source_name
HEK-based cell line
cell line
Epi-CTR
chip antibody
Lsd1 (Abcam, catalog# ab195405, lot# GR319278-1)
fraction
IP DNA

Sequenced DNA Library

library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
First, cells were fixed for 10 min at 37°C by adding room temperature Crosslinking Buffer to culture medium (to final concentrations of 50 mM TrisHCl pH 8, 100 mM NaCl, 1 mM EDTA, 1% formaldehyde). Fixation was topped by adding Glycine to a final concentration of 250 mM and incubates 5 min at room temperature. After two washes of ice-cold PBS, cells were scraped twice in 30 ml and 15 ml PBS. Cells were pelleted for 10 min at 4000 rpm 4°C, washed in ice-cold PBS and resuspended in 10 ml Cell Lysis Buffer (10 mM TrisHCl pH 8, 25 mM KCl, 0.5% NP-40) supplemented with AEBSF. After incubating 10 min on ice, lysate was passed 10 times through a 20G needle and incubated again for 10 min on ice. Nuclei were pelleted for 15 min at 3500 rpm 4°C and resuspended in 3.4 ml Sonication Buffer (50 mM TrisHCl pH 8, 10 mM EDTA, 0.1% SDS) supplemented with AEBSF. Nuclei were then divided in 2 tubes and sonicated for 20 min at 4°C on the Diagenode Bioruptor Pico according to manufacturer's instructions. Finally, poorly sonicated chromatin was pelleted for 10 min at 16 000g and supernatant was recovered and added to blocked Protein A beads (Ademtech, 04230) for pre-clearing of chromatin. After incubating for 3 hours at 4°C on a rotating wheel, 200 µl were taken as Input. The rest of the chromatin was incubated overnight at 4°C on a rotating wheel with IP antibodies: Lsd1 (Abcam ab195405). The next day, 50µl blocked protein A beads were added to the chromatin and incubated 4 hours at 4°C on a rotating wheel. They were then washed twice in FA150 buffer (50 mM HEPES pH 7.4, 1 mM EDTA, 1% Triton X-100, 150 mM NaCl, 0.1% NA-Deoxycholate, 0.1% SDS), twice in FA500 (50 mM HEPES pH 7.4, 1 mM EDTA, 1% Triton X-100, 500 mM NaCl, 0.1% Na-Deoxycholate, 0.1% SDS) and once in TE (10 mM TrisHCl pH 8, 1 mM EDTA). To elute chromatin, beads were resuspended in 100 µl ChIP Elution Buffer (50 mM TrisHCl pH 7.4, 10 mM EDTAs 1% SDS) and incubated for 1 hour at 65°C with 650 rpm shaking. Supernatant was then recovered, NaCl was added to a final concentration of 200 mM and incubated overnight in a 65°C waterbath to remove crosslinks. The next day, samples were treated with RNase for one hour at 37°C and with proteinase K for one hour at 45°C. DNA was then purified using Qiagen PCR Purification columns and quantified using Qubit dsDNA HS kit. ChIP-seq libraries were prepared with TruSeq ChIP Sample Prep Kit (IP-202-1012, Illumina)

Sequencing Platform

instrument_model
Illumina HiSeq 2500

hg38

Number of total reads
45743524
Reads aligned (%)
87.5
Duplicates removed (%)
26.0
Number of peaks
2806 (qval < 1E-05)

hg19

Number of total reads
45743524
Reads aligned (%)
85.9
Duplicates removed (%)
28.7
Number of peaks
1830 (qval < 1E-05)

Base call quality data from DBCLS SRA